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O R I G I N A L P A P E R

Preparation and characterization of chitosan

hydroxyapatiteglycopolymer/Cloisite 30 Bnanocomposite for biomedical applications

Amal Amin1

Heba Kandil1

Hanem M. Awad2

Mohamed Nader Ismail1

Received: 24 September 2014 / Revised: 13 January 2015 / Accepted: 3 March 2015 Springer-Verlag Berlin Heidelberg 2015

Abstract A new composite was synthesized from chitosan/hydroxyapatite and

glycopolymer grafted onto a Cloisite 30B clay surface (CS/HAP/clay-Gh) in

aqueous media via a solvent casting and evaporation method. Another composite

containing chitosan/hydroxyapatite and pristine Cloisite 30B (CS/HAP/clay) was

prepared and compared with the (CS/HAP/clay-Gh). The resulting composites were

characterized using various analytical tools such as Fourier transform infrared

spectroscopy (FTIR), X-ray diffraction (XRD), thermal gravimetric analyses (TGA)and scanning electron microscopy (SEM). Water uptake capability or swelling was

tested for each composite. The cytotoxicity of the resulting tri-component com-

posites was tested against a breast carcinoma cell line (MCF-7), liver carcinoma cell

line (HepG-2) and a normal human skin fibroblast cell line (BJ-1) by MTT [3-(4,

5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide] as well as LDH

(lactate dehydrogenase) assay. The obtained preliminary results showed good

properties for CS/HAP/clay-Gh on the way to be used in the future in the biomedical

applications.

Keywords Nanocomposites Glycopolymers Chitosan Hydroxyapatite Biomedical applications

& Amal Aminaamin_07@yahoo.com

1 Polymers and Pigments Department, National Research Centre, Dokki, Giza, Egypt

2 Department of Tanning Materials and Leather Technology, National Research Centre, Dokki,

Giza, Egypt

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Introduction

Tissue engineering could be an alternative strategy to renew the damaged human

organs and improve the quality of life of the patient where it has been used to

develop scaffolds to regenerate various tissues such as skin, liver, nerve, bone andblood vessels. [1] The success of tissue-engineered products mainly depends upon

the development of degradable extracellular matrix analogs that provide a temporal

support for cell adhesion, proliferation, maturation and differentiation. Natural

polymers such as collagen, chitosan, fibronectin, gelatin and synthetic polymers

such as polyesters and polypeptides have been used to improve the hepatocyte cell

functionality where porous scaffolds with high inter-pore connectivity are desirable

for the culture of hepatocytes [2, 3].

Accordingly, great attention has been paid recently to the biomimetic approaches

to obtain bioinorganic and organic composite materials [4]. Natural bone is acomplex of inorganicorganic nanocomposite materials, in which hydroxyapatite

[HAP, Ca10(PO4)6(OH)2] nanocrystallites are well organized within self-assembled

collagen fibrils [5] where the inorganic phase and a polymer phase are essentially

insoluble in each other [6]. There have been many attempts to prepare bone-like

biocomposites of HAP with several bioactive organic polymeric components such

as collagen, chondroitin sulfate, chitosan, starch, chitin, amphiphilic peptide [79],

poly(lactic acid) [10, 11] and polyamides [12, 13]. Chitosan (CS) and hydrox-

yapatite (HAP) are the best bioactive materials in bone tissue engineering where

HAP is the major inorganic compound in mammalian hard tissue because of thebiocompatibility and osteoconductivity [14], while CS is characterized by the high

biocompatibility, biodegradability, porous structure, osteoconduction and intrinsic

antibacterial nature [15]. Therefore, the composite scaffolds of CS with HAP were

extensively studied for bone grafting and other tissue engineering applications [16].

However, in spite of its potential use as scaffolds in bone tissue engineering, [CS/

HAP] composite scaffold has rather poor rheological properties because CS

undergoes extensive swelling in water which impedes the use of CS/HAP composite

scaffolds in load-bearing applications [17]. Thus, various components have been

combined with CS/HAP to form effective systems for tissue engineering such ascollagen, gelatin, poly (methyl methacrylate), poly (L-lactide acid), polycaprolac-

tone, carboxymethyl cellulose, carbon nanotube and montmorillonite [15,1824].

Significant enhancements in nanomechanical and thermal properties were

exhibited in case of [CS/HAP/montmorillonite (MMT)] scaffold compared with

(CS/HAP) composite scaffold [22]. That was attributed to the high surface area and

large aspect ratio of MMT as filler with HAP to reinforce chitosan and enhance the

mechanical properties and thermal stability of the resulting composite.

Furthermore, porous hydroxyapatite (HA) disks have been seeded with rat

hepatocytes into the peritoneal cavity of Nagase Analbuminemia rats. [25]

Angiogenesis and hepatocyte viability were maintained for at least 3 weeks.

Human hepatoma cell line BEL-7402 was cultured and treated with HA

nanoparticles at various concentrations. It was found that hydroxyapatite nanopar-

ticles inhibited human hepatoma BEL-7402 cell growth and induced apoptosis,

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which may add a new way to use HA nanoparticles for tumor treatment [26].

Different combinatorial factors like porosity, scaffold architecture, biocompatibility,

biodegradability with non-toxic degradative products and controlled release profile

will provide necessary signaling cues to retain liver-specific functions.

The aim of the current work was to develop some innovative materials that canbe used as scaffolds for the sustained growth of cells which may be involved in the

future in the regenerative medicine and tissue engineering, because they can be

potentially tailored to mimic the natural extracellular matrix in terms of structure,

chemical composition and mechanical properties. Accordingly, glycopolymer was

used as biocompatible polymer to modify the clay surface (e.g., Cloisite 30 B) via

surface-initiated atom transfer radical polymerization (SI-ATRP). Then, the effect

of grafting glycopolymer onto Cloisite 30 B on the properties of the resulting

composite was studied. Generally, glycopolymer is a synthetic polymer carrying

carbohydrate (sugar) moieties as pendant or terminal groups with importantpharmacological and biological properties mainly because of its hydrophilic nature

[27,28]. SI-ATRP has been used to prepare well-defined polymers with interesting

functionalities and architectures in addition to its ability to simultaneously grow

chains from multifunctional cores or surfaces such as flat wafers, particles, colloids

and polymers [29]. SI-ATRP is one of the best controlled/living surface-initiated

polymerization techniques because of its tolerance to several reaction conditions in

addition to relative easy preparative methodologies compared to others where SI-

ATRP is performed by an in situ surface-initiated polymerization from immobilized

ATRP initiator onto a solid surface [30].

Materials and methods

Materials

Cloisite 30B [31] was kindly supplied from Southern Clay Products, Inc. (Gonzales,

TX) with cation-exchange capacity (CEC) as 90 meq/100 g. Chitosan was provided

from Aldrich with degree of deacetylation greater than 75 %. All other chemicals

were provided from SigmaAldrich. Roswell Park Memorial Institute (RPMI) 1640

medium and Dulbeccos Modified Eagles Medium (DMEM) were purchased from

Sigma Chem. Co. (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased

from Gibco, UK.

Preparative methods

Preparation of glycomonomer (G)

The glycomonomer (G) was synthesized according to the previously reported

method [32] via esterification reaction of 1, 2: 5, 6-di-O-isopropylidene-a-D-

glucofuranose (38.4 mmol, 10 g) with methacrylic anhydride (67.1 mmol, 10 ml) in

the presence of absolute pyridine (50 ml) at room temperature.

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Grafting of glycopolymer (G) onto Cloisite 30 B clay surface via SI-ATRP

The nanocomposite was prepared according to the reported method by Amin et al.

[33] where the ATRP surface initiator (clayBr) was firstly prepared by treating

Cloisite 30B with 2-bromoisobutyryl bromide (BIBB, 150 mmol) in the presence oftriethylamine (Et3N) and anhydrous THF. Afterward, the prepared glycomonomer

was involved in controlled polymerization via SI-ATRP using clayBr where the

polymerization was carried using glycomonomer (G, 4 g, 4.9 9 10-2 mol), Clay

Br (0.2 g), 2, 20-bpy (0.0389 g, 9.8 9 10-4 mol) and Cu (I) Br (0.0179 g,

4.9 9 10-4 mol) in xylene at 90 C.

After certain time interval, the grafted G/clay (clay-G) nanocomposite was

separated from the reaction and hydrolyzed using formic acid (80 %) to regain the

hydroxyl groups in sugar rings in the grafted clay-G [34] where the OH-ended

polymer (clay-Gh) nanocomposite was obtained and characterized by FTIR, XRD,TGA and SEM.

Preparation of hydroxyapatite (HAP) [35]

HAP was prepared according to the wet precipitation method reported by Katti et al.

[35] by adding a liter of CaCl2 solution (19.9 mol) dropwise to a liter of Na2HPO4solution (11.9 mol) with continuous stirring. The solution was adjusted to 7.4 pH

using 1 N NaOH. The resulting solution was kept undisturbed for 24 h, then, HAP

precipitate was separated by centrifugation. The obtained precipitate was dried invacuum oven at 50 C for 48 h. The produced HAP was characterized by FTIR,

XRD, TGA and SEM.

Synthesis of chitosan/hydroxyapatite composite (CS/HAP)

0.5 g (50 wt%) of chitosan was dissolved in 38 ml of 1 % acetic acid solution. 0.5 g

(50 wt%) of HAP was dispersed in 10 ml of DI water. The HAP solution was added

dropwise to the chitosan solution, and the resulting suspension was continuously

stirred overnight to obtain a good dispersion of HAP in chitosan. Then, this solution

was poured into a Petri dish and dried in an oven at 80 C for 48 h to form thin sheet

of CS/HAP.

Synthesis of chitosan/hydroxyapatite/clay composite (CS/HAP/clay) [22]

Firstly, 0.5 g (50 %) from chitosan was dissolved in 38 ml of 1 % acetic acid

solution. Then, a suspension from 0.1 g (10 %) of clay in 15 ml H2O was added to

the chitosan solution followed by adding 0.4 g (40 %) of HAP precipitate. The

resulting suspension was stirred overnight to obtain a good dispersion of HAP in

chitosan. Then, it was poured into a Petri dish and dried in an oven at 80 C for 48 h

to form thin sheet of CS/HAP/clay.

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Synthesis of chitosan/hydroxyapatite/clay-Gh composite (CS/HAP/clay-Gh) [22]

The same procedure described above in case of (CS/HAP/clay) was followed here

but instead of using pristine Cloisite (clay), the glycopolymer grafted onto Cloisite

(clay-Gh) was used.

Preparation of pure chitosan sheet (CS)

It was prepared by the same way but without adding filler to compare its properties

with that of the resulting composites. CS was characterized using FTIR, XRD, TGA

and SEM.

Instrumentation

Infrared measurements were carried out on Jascow FTIR-430 series infrared

spectrophotometer equipped with KBr discs. Thermal gravimetric analyses (TGA)

were performed on TA Instruments Q600 SDT system with heating rate 10 C/min

under nitrogen flow. X-ray diffraction (XRD) patterns were obtained via Siemens

D5000 diffractometer equipped with an intrinsic germanium detector system.

Scanning electron microscopy (SEM) images were collected using JSM 5410,

JEOL, Japan.

Water uptake or swelling of the developed composites was performed according

to a former report [15] by determining the initial weights (Wdry) of the previouslyprepared sheets (CS, CS/HAP, CS/HAP/clay and CS/HAP/clay-Gh), then by

immersing them in distilled water for 24 h. Afterward, the sheets were gently

removed from the beaker, dried with a filter paper to remove the excess water from

the surface, and immediately weighed (Wwet). The water uptake (expressed as a

percentage) was calculated using the following equation:

Water uptake % Wwet Wdry

Wdry100;

where, Wwet and Wdry = the weights of wet and dry composites, respectively.

Cell culture

Breast carcinoma cell lines (MCF-7) and liver carcinoma cell lines (HepG-2) were

purchased from the American Type Culture Collection (Rockville, MD) and were

maintained in RPMI-1640 medium. Normal human skin fibroblast cell line (BJ-1)

was kindly supplied by Applied Research Sector, VACSERA-Egypt and was

maintained in DMEM medium. All media were supplemented with 10 % heat-

inactivated FBS, 100U/ml penicillin and 100U/ml streptomycin. The cells were

grown at 37

C in a humidified atmosphere of 5 % CO2. All experiments wereconducted in triplicate (n = 3).

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In vitro cytotoxicity activities

MTT cytotoxicity assay

The cytotoxicity activity against MCF-7, HepG2 and BJ-1 human cell lines wasestimated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-

mide (MTT) assay, which is based on the cleavage of the tetrazolium salt by

mitochondrial dehydrogenases in viable cells [36]. Cells were dispensed in a

24-well sterile microplate (2 9 105 cells/well) and incubated at 37 C with a disk of

5 mm diameter of each tested sheet or doxorubicin (positive control) for 48 h in a

serum-free medium prior to the MTT assay. After incubation, media and disks were

carefully removed, 40 ll of MTT (5 mg/ml) were added to each well and then

incubated for an additional 4 h. The purple formazan dye crystals were solubilized

by the addition of 200 ll of acidified isopropanol. The absorbance was measured at570 nm using a microplate ELISA reader (Biorad, USA). The relative cell viability

was expressed as the mean percentage of viable cells compared to the untreated

controlled cells [37].

Lactate dehydrogenase (LDH) assay

To determine the effect of each sheet on the membrane permeability in MCF-7,

HepG2 and BJ-1 cell lines, a lactate dehydrogenase (LDH) release assay was used

[36]. The cells were seeded in 24-well culture plates at a density of 29

10

5

cells/well in 500 ll volume and were allowed to grow for 18 h before treatment. After

treatment with a disk of 5 mm diameter of each tested sheet or doxorubicin (positive

control), the plates were incubated for 48 h, then, the disks were removed. The

supernatant (40 ll) was transferred to new 96-well sterile microplate to determine

LDH release and 6 % triton X-100 (40 ll) was added to the original plate for

determining the total LDH. An aliquot of 0.1 M potassium phosphate buffer

(100 ll, pH 7.5) containing 4.6 mM pyruvic acid was mixed to the supernatant

using repeated pipetting. Then, 0.1 M potassium phosphate buffer (100 ll, pH 7.5)

containing 0.4 mg/ml reduced b-NADH was added to the wells. The kinetic

changes were determined for 1 min using ELISA microplate reader at wavelength

340 nm. This procedure was repeated with 40 ll of the total cell lysate to determine

total LDH. The percentage of LDH release was determined by dividing the LDH

released into the media by the total LDH following cell lysate in the same well [37].

Results and discussions

Chitosan/hydroxyapatite/glycopolymer grafted-clay (e.g., Cloisite) nanocomposite

(CS/HAP/clay-Gh) has been prepared upon several steps according to Scheme1

where Cloisite 30B surface was firstly converted to ATRP surface initiator (clay

Br) by treating it with ATRP initiator (2-bromoisobutyryl bromide). Glycomonomer

(G) was prepared and characterized as mentioned in the literature [32]. Then, G was

polymerized via SI-ATRP using clayBr as surface initiator to form clay-G which

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was separated and purified from copper residues of ATRP. The clay-G was

subjected to hydrolysis reaction using formic acid where OH-ended polymer clay

nanocomposite (clay-Gh) evolved. The aim of the last step was to regain the

hydroxyl groups in clay-G for complete beneficial use of the resulting (clay-Gh)

nanocomposite. The resulting clay-Gh was then used in preparing the CS/HAP/

Clay-Gh composite. Afterward, three composites were prepared, one of them wasCS/HAP composite and the others were CS/HAP/clay and CS/HAP/clay-Gh

composites. The properties of the prepared composites were studied, especially the

composites containing glucose units, to identify the influence of its presence inside

the composites.

Thermal stability of the prepared composites

TGA was performed for CS, CS/HAP, CS/HAP/clay and CS/HAP/clay-Gh as

shown in (Fig.1) to reveal the thermal stability of the prepared composites. Fromthe TGA curves, it was observed that the onset points for the degradation of the

prepared composites were variable, i.e., the onset point for CS sheet was at 250 C,

while for CS/HAP, it increased to 265 C. The onset point increased to 270 and

272 C for CS/HAP/clay and CS/HAP/clay-Gh, respectively. The slight temperature

differences observed for the CS/HAP/clay and CS/HAP/clay-Gh composites might

be due to the presence of some covalent interactions occurred as in case of CS/

HAP/clay-Gh between clay-Gh and chitosan. The weight loss below 300 C was

attributed to the water evaporation and the loss of chitosan moieties.

At high temperatures as 500 C, chitosan sheet lost all its weight, whereas noweight loss was observed in case of CS/HAP, CS/HAP/clay and CS/HAP/clay-Gh

composites and that was ascribed to the presence of HAP which increased the

thermal stability of the composites. However, it was observed that the thermal

stability of CS/HAP/clay and CS/HAP/clay-Gh was slightly higher than that of

Scheme 1 Snthesis of CS/HAP/clay-Gh composite

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CS/HAP and this was attributed to, beside the presence of HAP, the existence of

clay platelets which played an important role in providing a thermal barrier and

delaying the weight loss of chitosan. The previous data indicated that the prepared

composites were highly stable at higher temperatures.

Chemical structures of the resulting composites

The chemical structures of HAP, clay, clay-Gh, CS film, CS/HAP, CS/HAP/clay

and CS/HAP/clay-Gh composites were identified by FT-IR analyses in the region of

4000400 cm-1 as shown in Figs. 2, 3. For HAP, the spectrum showed the

characteristic absorption bands of synthetic HAP at 1636, 1032, 962 and at

Fig. 2 FTIR of clay (a) and clay-Gh (b)

Fig. 1 TGA of (HAP) (a), CS/HAP/clay-Gh (b), CS/HAP/clay (c), CS/HAP (d) and CS film (e)

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564 cm-1 for phosphate group (PO4)-3 while the peaks of (CO3)

-2 appeared at

14641408 cm-1

and at 874 cm-1

but the hydroxyl group (OH) appeared at3447 cm-1. The FTIR spectrum of CS film (Fig.3A) showed the typical

characteristic absorption bands of chitosan where carbonyl group (C=O) appeared

at 1734 cm-1, but CH stretching and bending appeared at 1414 and 1379 cm-1,

respectively. Amine stretching frequency (NH) and pyranose (COC) stretching

mode appeared at 1145, 1077 and 1022 cm-1, respectively. Moreover, the

characteristic stretching frequency of clay (SiOSi) was observed at 1100 cm-1

and OH stretching frequency appeared at 3500 cm-1. All these characteristic peaks

appeared in FTIR spectra of the prepared composites (Fig. 3B), However, in the

spectrum of CS/HAP/clay-Gh composite, it was noticed that the peak at 3500 cm

-1

was very broad which was attributed to the presence of the hydroxyl groups in clay-

Gh which played an important role in forming hydrogen bonds between the

components of the composite.

Fig. 3 FTIR ofA HAP (a) and CS film (b),B CS/HAP (a), CS/HAP/clay (b) and CS/HAP/clay-Gh (c)

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X-ray diffraction studies

The XRD plots of clay, clay-Gh, CS film, HAP, CS/HAP, CS/HAP/clay and CS/

HAP/clay-Gh were recorded as shown in Figs. 4and 5. A distinct peak appeared in

case of clay (Fig.4) at (2h = 6.896; d= 18.2 A) where after grafting theglycopolymer, that peak disappeared which indicated that exfoliated structure was

obtained. XRD plots of HAP and CS films showed as in Fig. 4, the characteristic

peaks for both of HAP and CS where for CS film, its corresponding main peaks

were observed at 2h = 20 (maximum intensity) and 22.5, while the major

corresponding peaks of HAP appeared at 2h = 26.3, 30.1, 31.6, 32.3, 45.3 and 49.2.

In all XRD spectra of the three composites (i.e., CS/HAP, CS/HAP/clay and CS/

HAP/clay-Gh; Fig. 5), there were three observed diffraction peaks at 2h = 20.0,

26.3 and 32.3 which revealed the presence of both CS and HAP in the prepared

composites. In case of XRD spectra of CS/HAP/clay-Gh, it was observed that thedistinct peak of clay at 2h = 6.8 disappeared here which indicated that the clay was

well dispersed in chitosan matrix and exfoliated structure was obtained.

Fig. 4 XRD ofA clay and clay-Gh (b). B CS (a) and HAP (b, c)

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Morphology studies of the composites

The SEM images were illustrated as shown in Fig. 6for low and high magnification

pictures of CS, HAP, clay-Gh, CS/HAP, CS/HAP/clay and CS/HAP/clay-Gh.

Generally, in each case, the darker part represented the polymer, and the lighter part

was assigned to the fillers. According to the SEM images, HAP particles were

uniformly dispersed in the chitosan (Fig.6). Uniform distribution of HAP particles

and clay in the polymer matrix was observed which might be due to the electrically

charged nature of chitosan network.

Water uptake

The goal of preparing CS/HAP, CS/HAP/clay and CS/HAP/clay-Gh composites

was to apply them in the biomedical applications such as bone tissue engineering

and other bone repairing applications; so, the water uptake capability was tested

for each composite. It was evident from the results that adding HAP and clay

either treated or untreated to pure chitosan led to a significant decrease in the

degree of water absorption of chitosan. However, it was observed that the wateruptake in case of CS/HAP/clay-Gh was less than that in case of CS/HAP and CS/

HAP/clay (Fig. 7).

Fig. 5 XRD of CS/HAP (a), CS/HAP/clay (b) and CS/HAP/Clay-Gh (c)

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In vitro cytotoxicity activity

In this study, CS/HAP, CS/HAP/clay and CS/HAP/clay-Gh were examined in vitro

for their cytotoxicity activities against MCF-7, HepG2 and BJ-1 cell lines using

MTT assay, this assay aimed at measuring the percentage of the intact cells

compared to the control, as well as LDH assay to measure the cellular membrane

permeabilization (rupture) and severe irreversible cell damage (Table1). The

results for the three sheets did not show any significant cytotoxicity, after 48 h, in all

the three tested cell lines as they all showed very weak cytotoxicity activity ranged

from 5.2 to 17.5 %. CS/HAP/clay-Gh sheet showed the least toxic effect on all cell

Fig. 6 SEM of CS film (a), HAP (b), clay-Gh (c), CS/HAP (d), CS/HAP/clay (e) and CS/HAP/clay-Gh (f)

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lines. By visualized analysis of the cells under microscopy, it was observed that only

the area under each disk of these three sheets did not show any viable cells and this

was explained by the fact that each disk of these sheets covered the area under it and

therefore it prevented the media and the humidity as well as the CO2 to reach thisarea which resulted in the death of the cells only in these covered areas without any

toxic effect on the other cells. These results were in agreement with the previously

reported data which indicated that similar composite nanoparticles containing HAP

did not show significant toxicity where in one study, the in vitro biocompatibility

and the proliferation of bone marrow stromal cells (BMSCs) were evaluated on n-

CS/HAP composite membranes using MTT assay. Nevertheless, after 7 days, the

cells cultured with the composite membrane of 40 % n-HAP got the best

proliferation and the cells on pure CS membrane held the lowest proliferation

among the three investigated membrane groups (pure CS, 40 % n-HAP and 60 %n-HAP). The results showed that n-HA and its content had obvious effect on the

proliferation of BMSCs [38]. In addition, it has been reported that at 50 mg/L HAP

nanoparticles, no significant cytotoxicity was observed on MCF-7. However, at

higher concentrations, nano-HAP inhibited the proliferation of MCF-7 cells in a

Fig. 7 Water uptake of CS film (a), CS/HAP (b), CA/HAP/clay (c) and CS/HAP/clay-Gh (d)

Table 1 Measuring cytotoxicity of the prepared composites against MCF-7, HepG2 and BJ-1 cell lines

using MTT, LDH assay

Sheet (0.5 cm

diameter)

% Growth inhibition (% of control)

MCF-7 HepG2 BJ-1

MTT LDH MTT LDH MTT LDH

CS/HAP 15.9 2.1 16.5 1.9 12.4 3.8 13.8 1.9 13.5 2.4 14.8 2.7

CS/HAP/clay 17.5 1.7 15.8 2.5 17.2 1.4 17.8 .9 15.3 2.8 14.3 2.5

CS/HAP/clay-Gh 10.1 1.5 9.2 3.2 9.1 2.7 10.2 2.1 5.2 1.5 6.2 1.9

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dose-dependent manner. Induction of apoptosis might be accounted for the anti-

proliferation effect [39].

In addition, the biological response of pre-osteoblasts (MC3T3-E1) on the

nanocomposite scaffolds of medium-MW chitosan scaffolds with 0.5, 1 and 2 % n-

HAP was found to be superior in terms of improved cell attachment, higherproliferation, and well-spread morphology with respect to chitosan scaffold. In the

composite scaffolds, cell proliferation was about 1.5 times greater than pure

chitosan after 7 days of culture and beyond, as implied by qualitative analysis via

fluorescence microscopy and quantitative study through MTT assay. The observa-

tions related to well-developed structure morphology, physicochemical properties

and superior cytocompatibility suggested that chitosann-HAP porous scaffolds are

potential candidate materials for bone regeneration although it is necessary to

enhance the mechanical properties of the nanocomposite [40].

Furthermore, in another study, composite films were prepared from chitosan (CS)and gelatin (G) with the addition of HAP. The cell affinities were studied using

human osteosarcoma (SaOs-2) cell line. The cell proliferation and spreading of

Saos-2 cells were found to be higher for the films with high gelatin content and no

HAP; whereas for HAP-containing composites, higher cell affinities were obtained

for the samples which had high chitosan content (CS:HAP = 66.7:33.3 %) where

for 7 days, no toxicity and good proliferation were observed [41].

Finally, it has been reported that, greater cell viability and cell proliferation were

measured on MC3T3-E1 cell lines (osteoblast) treated with three kinds of HAP

nanoparticles (neutral, positive, and untreated), among which positively chargedHAP nanoparticles showed the strongest improvement for cell viability and cell

proliferation even after 7 days [42].

Conclusion

The rapidly developing tissue engineering field requires novel processing methods

and designs of scaffolds and membranes for guided tissue regeneration. Embedding

of HAP into polymeric matrices containing CS has gained much attraction since

HAP-type crystals form the main inorganic component of the bone structure and can

be used as supporting materials for tissue engineering. Therefore, CS/HAP/clay

composite has been recommended for bone regeneration and for other different

applications related to tissue engineering to regenerate various tissues of skin, liver,

nerve, bone and blood vessels by developing the suitable scaffolds for that.

Accordingly, CS/HAP/clay-Gh composite was prepared through solvent casting

and evaporation methods. FTIR, XRD, TGA, SEM, swelling measurements and

cytotoxicity test were applied to investigate the effect of grafting biocompatible

polymer onto clay surface on the properties of the resulting composite. The results

showed that the CS/HAP/clay-Gh tri-component composite is an extremely potent

class of biomaterials.

Also, the results of this study revealed that the composite containing glycopoly-

mer-modified Cloisite (i.e., chitosan/hydroxyapatite/clay-Gh) showed improved

thermal stability and no significant toxicity on all cell lines as compared to the

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composite with pristine Cloisite. Therefore, this composite could be used as a

suitable material for the delivery of bioactive compounds as well as tissue

engineering.

In addition, the in vitro results may provide useful information for the

investigations related to HAP composite nanoparticles for the applications in genedelivery and intracellular drug delivery. Such a composite membrane may provide a

good prospect for further research and development in biodegradable guided bone

regeneration (GBR) membrane for future applications as well.

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